Document jg18Gwr11BdJkOax2k2abGpZR
foes
R226 -- 359
SRPT T7001
MIN 315/022356
PERFLUOROOCTANESULFONYL FLUORIDE (POSF) T-Tebl>
IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST IN HUMAN LYMPHOCYTES
Sponsor 33MMCCeanrtpeorrt Toxicology SBiupilding T0250 UMsNa551333220
Page t0r3t
ResearchLaboratory WHouomilnlgydRoenaLidfe Sciences Ld AHocnoinnbguiroyn PCeaamstradkgesire ENGLAND Reporsued 22 Osober 2002
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CONTENTS
MIN3157022356 Page
COMPLIANCEWITHGOODLABORATORY PRACTICESTANDARDS...
4
QUALITYASSURANCESTATEMENT. ccm
eS
RESPONSIBLE PERSONNEL...
6
INTRODUCTION sss
8
EXPERIMENTALPROCEDURE...
HL
ASSESOSFMREESUNLTTS ccc
u--
ws 18
MAINTEONFARENCCOREDS...
rs smm--
15
CONCLUSION rss
2:
001543
sez
`COMPLIANCEWITHGOODLABORATORYPRACTICE STANDARDS
Ie ty dct se vs ceedi cmc ie lovin Ghoet Labor FE. ComisionDie PRoUf iEsh 590iorNo L770 OE RD pD ri ot Cou Ly Pre (ord 9)
tone whsrl cic 5sof hint, epre t e pi Teg eobe es wo pny ofeS,
;i fBye ltTxS opnas.a VeF_oe So Toi
Z2 other2m 2
) 001544
QUALITY ASSURANCE STATEMENT "The following have been inspeorcautdietedd in relation tothis sudy
MIN 3150022356
Study Phase
Date of Inspection
Date of Reporting
Protocol Audit
11 January 2002
11 January 2002
StudyBased Inspection Culture reament
17 January 2002
17 January 2002
Process Based Inspections Formulation Cultre Estaiishment Harvesting and slide preparation Side scoring 59 preparation
14 January 2002 11 February 2002 7February2002 5 March 2002 15 January 2002
14 January 2002 11 February 2002 8February 2002 March 2002 15 January 2002
Report Audit
15 April 2002
15 April 2002
DPirroetcotcoorlaAnuddCito;mpAannyauMdiatnoafgtehmeepnraottoicnodlicfaotredthsibsosvt.udy was conducted and reporid 10 the Study SSttuuddyyDBiarsecetdorIansnpdecCtoimopn:anAynMinaspenctiaonogfaeaipmnhdiaecsaetneodftahbiosves.tudy was conducted and reported 10 the aPrnodcreespseBiaisveedprIoncsepdeucrtieosnes:mpAltoyoerdaobnoutitsheytpiemoefthsistusdtyudwyeweascairnepdroogurte.ssTihnesspeectwieornes coofnrdouucttiende indreported to appropriate Company Managemenats indicated above cRoenpdourcttAeuddnitd: rTehpiosrtreedpotrotthhaesbSetednyauDdiiterd baeynthdceCotQmuapolaintryyAMssauranncae Dgeapeasrmitndmeiecnna.tteTdhisbsoavued.it vas "Trehseulmtest0hordesf,lepc trheroawcdaeanaddobuserrvaetiosns were found obeaccurately descnrdtiherbepeortded
``AGnrgoeulpaMlearhingsn, ager, BSc. M.Sc.FRB, MRQA.
Date
DHuenptairnmgednotn oLfiQuaSlciiteyncAesssuLrida.nce,
is
001545
RESPONSIBLE PERSONNEL
Linda Alls, DEA Tox., DES Pharm. Vet. France, SDteupdayrtDmiernecttoofr,Genetic Toxicology. Lincoln Pritchard, B.Sc., S`DteupdayrStumpeenrtvoifsoGre,netic Toxicology.
MIN 3150022356
6
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SUMMARY
MIN 3157022356.
A study was chromosomal
apebrfeorrmerdaitnto hiausosmeansns
ltyhempahboiclyittyesofcuPlteurrfelduoirnovoicturon.esulfony]
Fluoride
(POSF)
to
induce
Hphuymtaohnacrmlaygmgplhuotciyntiens,,andinexwphoosleed tobltohoedesctulsuurbes,tanwceerbeothsiimnulthaetepdresteoncediavniddeabbseynceadodiftiSo9n mioxf nrdivoefdthreoimncaubavtieornsperSiovde, cnelpdivsisioin wacs oarenstcedlussingwCeolecesmidge,rtehedc,ellsTwhaorhverstsed afnod sslihdees prepsotahatrmeetapdha,secels cobeuexalminded for chromosomal damage.
IInympohrodceyrtetso,
atshseesmsitotthiec
itonxdiecxitwyasofcaPlecrulfaltueodrofoocruanlelscuullftounryels
tFrleuaotreiddewit(hPOtShF)testtosucbuslttaunrceed
human and the
Solvent control. On the basisofthese data, th following concentrations were selected for metaphase
analysis:
First test `With and without S9 m-i3hoxurs treatment, 17 hours recovery: 1.252,.5and 5% viv atmosphere.
Second test
`Without 59 m-i20hoxurs continuoustreatment: 08, |and 2% v/v atmosphere.
`With$9mix 3hourstreatment, 17hours recovery:2,5and7.5% ivatmosphere.
Isnatbiosttchatlhlyeasbisgneinfciceaantndipncrreesaesneceinofth$e9mpriopxo,rPteirofnluoofromoecttaanpehsauslefofniyglurFelsuocroindtea(inPiOngSFc)hcraoumsoesdomnaol. aberrataitoanys dose level, whencomparedwiththe solvent control, ineitherest. dAosquealnetvielt,atiNvoesauntailsytsiicsaflolrpsoilgynpilfiociadnytwinacsreamsaedseiinthceuplrtuorpeosrttireoanteodfwpiotlyhptlhoeindecgealtlisvweceornetrseonl.nd highest Aalblerrpaonstiicevlels,codnetmroolnsctormapotuitnnhdegssecnasuistievdityloarfgethesutteissttiscyasltleymasingnditfhiecaenftfiicnaccryeoasfetshein$9thmeixp.roportion of ofticslcaosntcolguendiecd tahcattivtihteyteisnttshuibsstianncveirPoerfclyutoorgoeoncettaincestuelsftonsyylstFelmu,oruinddee(rPOtSheF)exhpaesrsimheonwtnalnocoenvdiidteinocnes: described.
a
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INTRODUCTION
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itauetpohnodmesecsrbeshsteudry odensisgnend aonasymepthshocaybsilcolfuPeedfliuncioroec.tnesafony Fucide (POSF) Th sty was condiunccomptlieancdewith oe fllowing guidelines
OMaEmCmDaliGauniCdeoimnosfoormetheAbTeersatiionng Tofs,ChGeumsidcealln.e 4(7139.97) Genetic Toxicology: In Viro ChUrSomEoPsAo(m1e9A96b)emHaeaglotnh TEefstf.ectEsPTAes7t12G-uiCd9el5i.ne2s2. OPPTS $10575 In Vitro Mammalian uo19mm75nnSileymtopdehoaons:. DTuhhnaifvsoertbsdeseacnhnidseKveeiddklbiaynadidsd1iy9np0ge0po.hfyTcshhaaysymafaeogrgchuiurnmiebnde(rPiHoAfs)yretoatrbhsue(cEdlvoaanorstaenGhdaitOdrReiernsdslasnin gh mice ich (owe 1960, 1oCbrsthaeins esmeteuddyi,aFnlo.loTldhoeianukeerafemoemwse.heeltiietnyuceimltalseledwnenrmhes.amopekriednsendatoonfmoePrtUaAwpahasbseepfooosrlibenedigantgnhdwedamitieedcdweiilthohhehst,eest vCi{oiealsmcte eoomsinodomfr .eestChhaht eerrcoamemolonysiy.oecmTeeehse<tiebimrneeafittonhcepsreemsammenotofayfpdamhapahmhseaeogscebctermelalasstyiinwobenewrhfleooelsteqthudbeuelnrcoioyendgxicassamluibunstheeeedqui feifnrpostrrt ocetlxhlliemddaiptvrsieesslioeyonncs1.ea3f-1eoI4rfn hours Tohnshteadprvessenpceerfaonrdmehdoenbtwioesnpaofr$e3cemciaxs.ionIs.nthTen hseecsontd etss,,acoemsinHoiuorusreessmeennvtatswiussesdeid hou mie,and he 0 wi5 hix vaaEps fhe rt st Aeobernmtecioxnecs shwearfodsmoormheedisqncuioracinnitgn oD1NoAhfe(ccHrleoalmslaiesfoacmnideonBshooefmadhieoynsS.C_19N70S,(o1S9ma8ey9,aEFprT,arkdBsihOo,WnEHlNlu,y1fgdaapesPabhtaeavnke t1e9n91caTiinesy te rol umber of els conning aberions both with and without gaps has Ma any suhtbhset.taasrnmcheosetedofoanunondtdpierxeecrstelnaacremeofaesag.SeupTphleeefrfmeeecfntotreudnhellcetlrhueyfeushtaoavenndb(tee5es9tnsmmuibexsa)tbaponrcleeipeawrdeerdbyfinrecounmbzayrtameteds aeonnolndeAnmeeds i19t8%h, Naatanracjaen t(lA.r1l9o76)1.359 Known 10 duce igh lve of nay civiy
.
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MIN 3157022356.
"T`ohne3p1roAtuogcuoslwt2a0s0a1papnrdobvyetdbeyStHuundtyiDnigrdoencLtiofenrS1c0ieJnacneusarMya2n0a0g2ement on 18 July2001,by the Sponsor `The study was conducted at Huntingdon Life Sciences Lid, Eye, Suffolk, [P23 7PX, England. Trehsepecetxipveerlyi.mental star and completion datesofthe study were 15 January 2002 and 15 March 2002,
9
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Identity: CAS No: Appearance: Storage conditions: Batch number Expiry:
Purity: Date received:
`TEST SUBSTANCE
MIN 3150022356
Pefluorooctanesulfonyl Fluoride (POSF) 307357 Clear liquid Room temperature 040227 `Sponsor's responsibilty; assumed stable or the durationofthe study 955% 14 June 2001
or
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EXPERIMENTAL PROCEDURE
MIN 315022356
CULTURE OF LYMPHOCYTES RHuEmMaIn1b6l4o0odsvuaes ccoulllteucrteedmseedpiiucmllsyupfprloemmehnetaeldhyw,intohn-1s0m%okfioncgalmlceafdosneorrusm,,po|oulendiavnd dHielpuatreidn,it2h0 LmedUiumpe0n.i1cimllinp?h20yoghiane]masggelpctioimnyonf taendcl2l0sumspMensgilountawmeirnee.plaAcleidqunotsste(l0e4unmilverbslaolodco:nt4ai5nemrls athnedcienlclubated a 37C for spproximatly 48 hours. The cultures were gently shaken daly oresuspend
POSITIVE CONTROLS Inthe absence of$9 mix dSveppnioyr BApaptecahrnaunmcbe:er Sabvent:
Final concentration:
Inthe presenceof $9 mix MSuepnplii:er Appearance: SoBlavtechntnumber:
Final concentration:
MSiigtnoamCeheimniCcal CoLid B2lu0e p5o0wer Sterile purifidwater
0.2 pg/ml (3hourtreatment) 0.1 pg/ml (continuous treatment)
CnyicalMoepdhiscpahLaniide Whicpowder OSeNr4l6e5pu(rTiefsited1)vaantder1H485(Test2)
10 pg/ml
PREPARATION OF$9 FRACTION
SoSpxaces
MRaale
SSouusrice:
SCphraargeuseRDiavweireUyKderived
pWeyigh:
7SoBwoepsks
"
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MIN 3150022356 $li9vefrsacwteiroen swtaismuplrateepdarbeydAfrroocmloarg1r2o5u4,poafdmican.is1t0eraendiamsal.sinMgilexeindtafpuenrtiitoonneaolxiidnajsecetisoynstienmcsoimn itlheartaa daotssawgeeorefkSi0ll0edmga/nkdgthbiordyvweerigshtas.epOtincatlhleyfriefmthovdeady.after injection. following an overnight sarvation, the T0h1e5fMolKlCoIwi(n3smtiepKs Iwe:re1gcalrievedr)obuetfoare0be-4ingCtrunadnersafseeptrotieacdncohnodimtoigoenns.iseT.heFollivleorwsinwgerperepplaarcaetidoni,n hdiespheonmsoegdeinnaoteasliwqeuortescaenndtrsiofurgeeddaat 89000C0orfboerl1o0wmuinniulterse.quiTrehd.supernatant faction (59 fraction) was
PREPARATION OF $9 MIX P59H7m.i4x1co0n0aimnMe)d,: g$l9cforsaect-i6opnh(o1sp0h%atvAe)(.5MmgMC),lN(8AmDMP).(4KmCbI.(33AmlM)th,e scoofdaicutmorosrwtheorpehofislpehra-tsetebruiflfsecrd befor us.
TREATMENT OF CELLS WITHTEST SUBSTANCE - FIRST TEST Acufleturreapmperdoixuimma.teAltym4o8sphhoeurress,otfhPecruflltuuroersoowcearneescuelnftorniyflugFelduoarniddeth(ePcOeSlFls) wweerree reesstaubslpiesnhdeeddiinsefsrleesdh `2l0a,s4s0baontd7s0(%16v0ivmiatimnotsepmhaelrev.oAluimre)wawsiwtihtshedprtauwmnfcarposm0eagcihvbofirnaelacnodnctehnetnraatiaopnpsroofpr1i.a2t5e.v2o3l,u5m,e1o0f, Pheersfueopruomoscaapn.esAafftoernyevFaipoorraitdieon(oPfOPSeEf)lwuoarsoienccaondcuscuefdonuyilnFgluaosryirdien(gePOaSnPd)naenedde,quiilniebrraetdiotnhorfotuhgeh athtemnosipnhceurbeasteadt o3n7 thCe,itshiedelsymatph3o7cyCteicnualtruorlelserwaeprpeariantjuesct(esdeeinaopptehnedibxol3:e.AppTahraetgulsas{sorbovtalepsowuerrse paphparsoexiemxaptoelsyu.reTohfecullytmipvhatoecdytmeasmcmoaatlitahen icneslild)e,owfhthiechbororteastesantdhewebraeteismmoenrcseedevienrcyuletiugrhet mmienduituems `hoencreevvoelrutyiorne.volTuhtieonsoalnvdenetxpcoosnetdoldir(eActkl)y twoaPserefstuaobrloioscheadneisnufdounpyli!caFtleuocruildteur(ePsOSaFn)d fcoorntthaeinreedst aonf amnodspashoerceonoafinsiesd. aMniattommoyscpihnerCe,oaftaairfinal concentration of 02 gm, was added to duplicate culures cIumlmteurdeisaenldydibsecfaorrdeedt.reaTthmiesntwoafstrheeplsaecceodndwistehto1fmcuiltoufreSs9, m1ixm.lTohfemceudltiuurmeswwaesreretmhoevneadddferdom(0estchhe acopnptrrooplricsuelgelassswbeoruelecgsiivaiblnigsthheed suanmdeersarnsaomfofsipnbalerceoncoefntaria,tionCsycalsoapbhoovse,phTamhieddeupwlaicsataedsdoeldventto duplicate culturesa a final concentration of 10g/ml that were contained in an aimosphereofair. aTnhdereeshuosuprsenadefdeindofsriensgh,mtehedicuulmtuurnedsewrerane tcemntorisfupgehdoeaftariSe0r0in ufnoirve5rsmailnuctoens.tainTehrse.ceTlhlseyweweereritnhseend incubated for further 17 hours.
in:
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MIN 3150022356
HARVESTING AND FIXATION T(Swiogmhao)urtso ebaecfhorcatlhluerecelaltsawefirnealhcaornvcesetnetdr,atmioinotfe0.ac1tivpigtym.wasAafrteerst2edhobuyrsaddiintcuiboantioofn,Coelaccehmciedll Swuesptentsreiaotnedwawsitthranhsyfpeorteodntiocascoelnuttriiofnu(g0e0tu7b5eManKCIcepnrteriwfaurgmededfoartS37miCn)u.teAs aft eSa01g00.miTnhueteceplerpieoldleotsf Hpyeplotsofnicxeidncbuybaatdidointioant o3f7rCe,htlhy psruesppaernesdiocnosldwfeirxeaticveea(i3iupagnesd matetSh0a0nogl :fo1 pSatmignluatcieasl aancedtitchecicde)l. "Thefixativews replaced urer times unlitbecame colourless.
SLIDE PREPARATION "sTmhaellpevlolleutmweeorfe frreessuhspfiexnadteidve,.thAenfcoewntdiroupgseodfathe50c0egl. sfuosrpensmiionnustewseraenddrfionpaplelyd roenstuospperned<eedainncd3 pmriecpraorsecdopien bsulifdfeesrewdhiwcathewre(roeHt6h.e8n).alAlfotweerdri0nsiainrg-dirny.bufTehreedsliwdaetserwetrhe stihdeensswtaeirneedeifn t10s%rGriyemasnad. cfioemrplmeotuendted in DPX. The remaining cules in fative were stored at C until side analysis was
MICROSCOPIC EXAMINATION "Tmhietporicepcaerllesd pselride1s0w0e0rceeelsxianmeiacnbhyelcuilgthutremwiascrrecoordusesdicnegoxacpelpotywfoprowpoesritoibvjeectciovnet.olThtereaptreodpocrutlituorneos.f SForlovmentthceosnetrroelsuvlatlsutehoerdoifsethelreveelwacsaunsoindgecaredaesce,retashemianxmiimtucimcacinhdieexvabolfeacpopnrcoexnitmraatteiloyn wSa0s%uosfdth8e5 hseelechtiegd.hest dose level for the metaphase analysis. The intermediate and ow dose levels were also "dTohseecdonucnleenstsratiporneloifamicnahrypsoesatniovfemceonttarpohlacseomfpigouurnesdisnedlieccatteeddfoan iannsaulfyfsiicsiewntsletvheelolfowbeesrtrcaonntcecnetsra.tion e"Txhaemsienleedctesdt salimdaesgnwiefriecatthieonn coofde1d.00M0etuaspihnagseanceGlills wimemrerisdieonntifoibejdecutsivien.g Olnowe phouwnedrreodbjemcettiavpehaasned: iSghouwriengwearehiegxhamlienveeld,ofwhsebreerraponstsicbellles,. froCmhreoamcohscoumleurea.berrTahtiisonsnuwmebreer swcaosrerdedauccceodrdiinngculturtehse nCldaosrseifdiucpaltiiocnatoefdthceelIsSCwNer(e19n8o5t).ed Ownhleyncseelnl.wiTht#eh4ve- n48icechrreoamdionsgosmoefsawlerbeearnralaytsemde.tPaoplhyapsleoifdigiunreds were recorded. "fTohreneignactiidveencceonotfroplocluylptluorieds amnetdacpuhlasueescetlrles,atoeudtwoifthS0t0hemheitgahpehsatdseosceellvs,ewlaofstdehfefresitnseudbsqtuaanncieautsievdeliyn theanalysis for chromosomal aberations. "vTahleuenuumsibnegsoFifshaebre'rsreasntt m(Feitsahpehra1s9e73c)l.l in each eatment grwaoscoumpapredwth hesolvent contol
el
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SECOND TEST
tCruelattumreenstwwaerseusineidtiiantetdheanadbsmeanicnetoafinSe9d maisx.prIevnitohuselpyredseescnrcieboedf. S9Imnixth,isatsherceoenhdotuersttraeactomnetnitnuwoauss used,as inthefirsttest. Theharvesttime wasat20hoursforbothparts ofthetest. Concentratioonfs `Perluorooctanesulfonyl Fluoride (POSF) were as follows:
WithoutS9mix: 0.1,02,04,06.08,1, 24nd 5% v/vatmosphere.
With $9mix:
06,0a8nd.7.51%v/,vat2mos,phe5re.
Duplicate cultures were used for each treatment and concentration of 0.1 ug/ml, and Cyclophosphamide, aa
the solvent control. final concentration
Mitomycin of 10 g/ml,
C, at a final were added to
duplicate cultures.
Three hours resuspended
after dosing, the cultures in fresh medium under
containing S9 an atmosphere
mix were of air in
centrifuged. The cells universal containers.
were They
rinsed were
and then
incubated foar futher 17 hours. Cultures treated in the absence of S9 mix were incubatedfor20 hours.
Al end
cultures were treated with of the incubation period.
Colcemid, They were
at a then
final concentrationof0.1 pg/ml, two hours harvested, fixed and the slides prepared as
before the previously
described. The slides were then examined microscopically as previously described.
STABILITY, HOMOGENEITY AND FORMULATION ANALYSIS Tdehteersmtaibnieldityasapnadrthoofmotgheisnesitutdyy.ofAtnhaelytsesits soufbsatcahniceeveadncdonocfentthreatteisotnswuabsstnaontcpeeirnfothremesdolvaespnatrwtoerfetnhoits study.
1
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ASSESSMENT OF RESULTS
MIN 3151022356
cAunrreanstsahyistisorciocanlsicdoenrtreodltroanbgee.acceptable if the negative and positive contol values le within the "The test substance is consideredtocause apositive response ifth following conditionsare met:
cSthartiosmtoicsaollmyessig(neixfcilcuadnitnigngcraepass)erse(Po<b0s.e0r1v)eidntahteonfereoqrumeoncryeotfesmtectoanpcheantsreastiwoint.h aberrant "The increases exceed the negative control rangeofthis laboratory, taken at the 99% confidence limit
`Theincreasesare reproducible between replicate cultures. "The increases are not associated with large changes in osmolality ofth treatment medium or extreme oricity.
Evidence of a doserelationship is considered (0 support the conclusion. aAbonevgeactoinvceurrersepnotncsoentirsocllafriemqeudienfcnieosasraetoibstsiecravleyds,igantiafnicyadnotsienlcerveeals.es in the numberof aberrant cells A further evaluation may be carried out if the above criteria fora positive or a negalive response are not met.
MAINTENANCE OF RECORDS All raw data, samples and specimens (if appropriate) arising from the performance of this study will remain the propertyofthe Sponsor. Taynpdeasrcohfisvainmgplmeaayndbespdeicsipmoesnedwhoifchaftaerre tuhnesuiptearbiloed,sbsyatreeadsoinn oHfunintsitnabgidloitny.LfiofrelSocnigentceersm Srettaenndtairodn Operating Procedures. aAlrlchoitvheerfosramappleersioadndofspfeicveimyeenasrsanfdroamll tahewddaatteaownilwlhbiechretthaeineSdtubdyy HDuinrteictnogrdosnigLnisfethSecifeinncaesreipnoritts r`eAftteenrtisounchotfimteh,ethmeatSeprioanlss.or wIifllrbeequceosntteadc,teHdunantdinhgidsoandvLiicfeesSociuenogcenshthtweilrletcuomn,tidniusepostaolroertfiunthtehre materials subject 0 reasonable fee being agreed with the Sponsor. oHfutnhteinfgidnoanreLpiofretSicinesncaerscwhiilvlerinedaeifnintitheelyQ.uality Assurance records relevant to this study and a copy
15:
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RESULTS
MIN 3157022356
FIRSTTEST
Toxicity data MarietosthiocwinndnicTesabolfec2u.ltured human lymphocytes trated with Perflucrooctanesulfonyl Fluoride (POSF) Iinndtehxetaob5s7e7nc%eofofthSe9somlivse,ntPecronftlruoolrovoacltuaeneastudlofsoneyllevFelluoorfi5de%(vPiOvSaFt)mocsapuhesreed.a Trehdeucdtoisoenlienvetlhsesmeilteocttiecd forthe metaphaseanalysiswere 1.25, 2.520d 5%vivatmosphere. iInndtehxetpor5es2e%ncoefotfhe$9somlviexn,tPceornftlruoolrovoacltuaenaetsudlofsoenyllevFelluoofri5d%e (vP/vOSaEum)ocspahuesreed.aTrhedeudctoisoenlienvetlhsesemliteocttiecd forthe metaphaseanalysiswere 1.25, 2. nd 5%vivatmosphere. `fTihgeureqsuawnhietnaicvoempaanraleydtsiosthfoerspoollvyepnltociodnytrsolh.owed no increase in the number of polyploid metaphase
Metaphase analysis
TlheyemffepctshaorfoesPcherofwylnuiotrnoToeacubaslnees3ualfnodnys]umFmlauroriisdeed.
(POSF) in Table
on 1
the
chromosomes
of
cultured.
human
sInuabtiostthcatlhye asbigsneinfciceanatndintchreeapsreesinentcheeopfro$p9ormtiixo,n PoefrcfellulosrowoictuhnecshurlofmoonsylomFalluoarbiedrera(tPiOonSsF)atcaaunsyeddonseo level, when compared with the solvent control.
sBiogtnhifipcoasnttiivnecrecaosnetrso(lP<c0o.m0p0o1u)nidnst,heMpirtoopomrytciionn oCf aabnedraCnytccleolplh.osTphhiasmdiedmeo,nsctaruasteedd tlhaergeeffiscaatcsytoifctalhley `59 mix and the sensitivityofthe est system.
SECOND TEST Toxicity data MarietosthiocwinndiinceTsaobfl4ceultured human lymphocytes treated with Perfluorooctanesulfonyl Fluoride (POS) Iinndtehxetaob6s1e9n%coefotfhSe9somlivxe,ntPceorfnltuoolrovoacltuaeneastudlofsoenyl]evFelluoorfi2de%(vPiOvSaFt)mocsapuhseerde.a Trehdeucdtoisoenlienvetlhsesmeilteocttiecd forthe metaphase analysis were 0.,1 and 2% viv atmosphere.
16:
001556
MIN 315022356 Iinndtehxe tpore3s9en%coefothfe9somlivxe,ntPecronftlruooloovcatlaueneastudfoosneyleFvleuloroifd7e.(5P%OSviFv). cTahuseeddoasereldeuvcetlisosnelinectthed mfiotrotthiec metaphase analysis were 2,5 and 7.5% viv. "wThheenqucaonmipaatrievdetaontahlyssioslfvoernptocloynptlrooli.dy showed no increas inthe number of polyploid metaphase cells Metaphase analysis Tlhyempehffoecctystaeroefs PsehrofwlnvoirnooTcaanebsulalfneodnysl uFlmuomriader. ni(PTsOaSebFld)e 1on the chromosomes of cultured human Isnuabiostichatlhley asibgsneinfciceanatndintchreeapsresienntche oprfo9pormtiixo,n PoefrfcleulosrowoiclthanceshurlofmoonsylomFalluoarbiederr(atPiOoSnF)atcaanusyeddosneo. level, when compared with thesolvent control Bsiogtnhifipcoasnittiivnecrceoansterso(lP<c0o.m0p0o1u)nidst,heMpirtoopmoyrtciionnoCf aabnedrraCnytccleollpsh.osphamide, caused large, statistically
CONCLUSION
1ofsclcaosntcolguedniecd tahcaivtihty tiensthsiusbstianncveirPoerfclyutoorgoeoncettaincestuelstfonsyy! Fsl.uoruinddee(rPOthSeF)exhpaesrismheonwtnanl ocoenvdiidteinocnes described.
7:
001557
REFERENCES
MIN 3150022356
EVANS, HJ. and O'RIORDAN, M.L. (1975) Human peripheral blood chromosome aberrations in mutagen tests. Mutation Research, 31, 135
lymphocytes
for
theanalysis
of
FISHER, RA. (1973)TheExact Treatmentof HafnerPublishingCompany, New York.
2
x2
Table
in:
SarisicalMethods
forResearch
Workers.
GBRAALDLLOEWYA,YM,OS.M(.198D7)EAESfYf, eoDfcAhti.gshBoEsAmNot,icCsLtr.engKthRoAnGcNhrAoKm,osAoRme.abAerRrMatSioTnRs,OsNisGte,r cMhJro.mataindd exchanges and DNA strandbreaks,and the elation to toxicity. Mutation Research, 189, 15.
HEDDLE, JA. and BODYCOTDE.J,. (1970) On the formationofchromosomal aberrations. Mulation Research,, 117,
ISCN (1985) An KLINGERH,. P.
Intemational System for Human (Eds). S. Karger AG, Basel.
Cytogenetic
Nomenclature,
HARNDEN,
DG.
and
MMAutRaOtiNon,RDesMe.arcahn,d 1A1M3E, S17,3.B. N. (1983). RevisedmethodsfortheSalmonella mutagenicitytest
NATARAJAN, AT., TATES, AD. van BUUL, PPW., MEUERS, M. and de VOGEL, N. (1976) `RCesyetaorgcehn,e3t7,e8ff3e.cts of mutagens/carcinogens after activation in a microsomal system in vitro. Mulation
NOWELL, P.C. (1960) Phytohaemaggluinin: leukocytes. Cancer Research 20,462.
an
initiator
of mitosis
in
cultures
of normal
human
SATYA-PRAKASH, K.L., HSU,T.C. and PATHAK, morphology. Cytogeneticsand Cell Genetics, 30, 248.
.
(1981)
Chromatd
lesions
and
chromatid
core.
SCOTT, D., DEAN, B.. aberration assays in vitro
inDAKNIFROKRDL,ANN.DDD..J,.a(nEdd)KIBaRsKiLc AMuNtDa,genDi.Jc.ity(1T9e9st0s):
MUetKaEpMhaSseRecchorommmoesnodmee.d
rPervoicseeddurpe.s6.2.RCeampboorfirdtghteeUUnKivEeMrsSitsyuPbr-escso,mmCiatmibreiodnge.guifdor emutlageniicintyteestsing. Report, Par|t
18
00155! 8
Testl Epxeproiosde (hous)
3
3s
TABLE1 `SumomfRaesrulyts
MIN3151022356
Pefs`oCroonocceunnteastuonlfoofnyl CeElxlcslwuitdhinabgegr3aptions | CelIlnsclwuidhinagbgearapisons | RMeiltaottiivce
Fluoride (POSF) (Govivamosphere) | Iodividal | Mean | Individual
Mean | Index
wes ) | ies () @)
oan 125
T1102 2 20 100 5 4 as |s os so
25
300 as [32 as n
s
s 1 30 |1 1 a0
02pm Mitomycin) [#18 26 200s *28 *28 2600
5 -
ocin 125
2 2 20 |e4 4 a0 100 voso20 [1s 30 9
25
30s a0 [3 6 as 0
5
ora [12 as 2
10ngm)Cyelopposphamite)| 20 23 mows [m2 29 267eee
Tea2
pEexrpoisoudre
(hows)
Perf`lCuoonrcoeonctuantcisoinfofony! CaElxscwhiutdhisnbgegraapisons| CellIsnwcihtihnsgbegraapisons | RMeiltaottiivce
Fluoride (POSF)
(5 viv mosphere) [ dvd" Vin] vIanlduievsid(u8al) M(e%)an In)dex
oan 08
2 0 10 |2 0 10 o 2 10 |o 3 us
100
21
2 1 as [31 20 & o 0 00 [0 o 00 a
OlugmMiomycinC)| 9 7 sowe| 9 7 some -
own
toro
[22 20 10
2
ror
32 as 7
5
o 1 es 12 as 0
25
oo 1 07 [0 2 13 ES
10pm(Cyclopposphamide)| *18 +18 1sovee [20 *20 20wee -
Otherwise 2P0<00010l
5500ccellllswweerreeaanaylyssed rroommttessccuurreesoeetoo sigh clevelseoafpsbharasisopnrseesteon de
19:
001559
MIN 3150022356
TABLE2
Mitotic indexdata - first test
`Without S9 mix, 3 hours treatment and 17 hours recovery
Concentration of Perluoroocunesulfonyl
Fluoride (POSF) (% viv atmosphe
0 (ain
Mioticindex# | Relaitnivdeexmi#totic
Incidence __%Mean )
1000 90
100
92/1000
Polypiowdy
Incidence %Mean
vs00
02
S00
125
511000 sa
51000
25
741000 [a
n
551000
5s
4S571100000 si
51
2500
02
500
10
a
0
bb
"
bb
Ek)
b
b
#3 CVearlycufleswomnsehatvebaeeppnrehasedanetsosniengsdreounded values b Nocels.no meaphases presenonside
120:
"
001560
MIN 3150022356
TABLE2
Mitotic index data - first test (continued)
`With $9 mi, 3hourstreatmentand17hoursrecovery
PerflCuoonrcoeoncmuanctsounlfoofnyl (5Flwuioriadmeo(sPpOhSePr)
o "wn
Mote index # | Reliivnedemxooie
Incidence %Mean
ome 99
100
1001000
Faiypondy Incidence %oMean s50o0o 02
125
1o1w371000 91
2
25
s93n10o00 79
s
2de0m1000000 si
2
0550000 00
0
a
2
b
"
bb
0
b
B5b NVCeorcayefllwsonnhovmehcalev.bameeonrpmapaprhdehesaeissnieonsengsrriossudnedeodnisliees: T Nomensd
a
001561
MNsis22356
TABL3E
[I -------- Wau 59mi,hs esterand 17 hors covey
reCPpoomemstornostsD |NT owe] FO omion |tTaobemedi_[Re--jlre
rs cooee||eom | fo en
w;n wwll
Vo| lwee]: ww
us wa|H
x w0| s s ww||
Ces ae sls so] @ : s|e ass|: wm 2 l:s slr: ww
oanroems || 00 | |B
i|Bmol m=o -
32@ I CEwme m
ooiBwRe
Cometem SEeE r pR ies
Snr
onwo rFoooom
2
001562
MIN 3150022356
TABLES
Metaphaseanalysisdata - rst est (continued)
With$9mix, 3hourswean:ad 1 hours covery
PeFrCrooonrcdeaean(ctOloSonDnly|c|xaNnoisnteld]|Coma [CRroomemsaoni?n
(% viv atmosphere)
@on
wtyopee. | obopeoe ww| |:
|Noolsen [RVieiinc]
:2 20|e: wo mw
12s
ww||
,
2s
ww| |3s 3
s
ww| [1
3L201s se] w ssals`a) mw Li owe 2 oas| =
rye 0
(Cyclophosphamide) 100
7a]
1 24
0 zo|u ar| -
23 ee |29 e-
@GGQS CCGmmErRtTeeteSG CGaoenmmmieecnmoomnde Th C CeE p teni s hea eds
ones r00o1m
5:
001563
MIN 3150022356
TABLE4
Mitotic index data - second test
`Without $9 mix, 20 hours continuous treatment
PerfClounocroecncaunieosnufoofnyl Wioieindox # | Reliivnederxao
Fl(uwoirvidseum(osPpOhSeFr)e) | Incidence __%Mean
o
nenoo 17
100
an
1071000
Palypody Incidence %oMean 50 0 0500
or
v1131i000 109
5
02
n103e110o00 109
0
04
s571u10o00 94
0
06
s64n10o0w0 85
08
0n10o00 81
'
80 11000 77
o
2
71e0n0o0 71
a
a5s00 00
s
2w010i00 22
1
& Caclaions hveben made ingrondedvies
24:
001564
MIN 3157022356
TABLE4
Mitoticindex data- second test (continued)
`Wit$h9mix, 3hours treatmeanntd 17hoursrecovery
Per"fClouonrcoearciurnacusounloffony! Monitor | Reliivnedemxio
Fluiovrideam(oPsOphSeFr)e) | tncidence 9% Mean )
o an
eno 82 941000
10
Polygon Incidence &Mean 550000 01
06
7m41i00o0 82
100
0s
0 7911000 76
E
'
s81e00o0 70
8
2
5a9710000 60
5
sS6m10o00 57
3
2s
0 39/1000 32
oonns
00
# Calusihavoebneesnmdewing rounded aes
25:
001565
Concemeatonal PeruFclruooorciudnee(sPaOlSfFo)ny!
(%viv atmosphere)
ioo
MIN315022356
TABLES
Metaanalyspisdaht -asecsondteest.
`Without 9mix, 20hourscontinuous esment
Remon| No orwenamas [Ree
romana [Gromoson| Gare [Bc ew]
Mdieoixc
obtypec. olorypea
og cs gps %
*
w0 | 1
2 10)2o 10] w
os
100
w| 2
|
w| 2
100
2
100
100
0 w0|o us| e 23
2 oas|a 20 a
1
1
0 oofo oof a o
01 ppm domyene)
|
w a0
|
3[6
23
To osollo soomf -
@SD Obenie
eCCmmmomwitmbrteekk rP2o00m1
cmew OOvrormsodeocumen GSGr CCrolvemaamnemmrseerrSneimnmseesr pbesed els
126:
001566
APPENDI2X
MIN 315022356
Historical positive controWlidatteh(Jo$a9numuiatxry 199-9 December 2001)
O xctinggaps,lower99% confidence i = 8.5%,mean= 17.12%
{Ehcludinggaps,lower 99% confidence limit = 10.0%,mean = 19.95%
LoEY
a
Sa
i
gE
5s
BIRITEHA
Eo s
fll | iBl
0
13
Hl
5 10 15 2 25 W IB
0 & 0 5 60 6
Frequency of aberrant metaphases (%)
Matorical positive controlWdiantsSJoamniuxary 1999- December 2001) Ecchigaps,owerS9%confidenceli = 7.0%,moan = 14.95% hold gas,ower99%confidence i 8.5%,moan = 18.40%
PF so
gia= Iis
5 0 5 10 1s m 25 % 3 0 45 0 5
Frequency of sberrant metaphases
2
001567
JR-- OAR SOR ReHeEE
Culture bottle (volume 160ml)
Cote apdr ams ------m--stcl
O
`Rotating bottle holder
O
30
| Motorised rollers 001568
he oi opt St 1 or, *RTay
& wm:
HEav
F-- E Eom-- ns
covuusonsonsracrce
ann EE STATEMENTOFCOMPLIANCE ere
BEEpA rain
sEEsppmaEacs,
H. E Mutagenicity
eR eerrereinecn T A Se um m
Be Jt In
RR
.
:
:
VU1569